Journal: Cellular and Molecular Life Sciences
Article Title: A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins
doi: 10.1007/s00018-023-05102-1
Figure Lengend Snippet: G4 recognizes soluble forms of CD13, with higher affinity and selectivity than bestatin, and the membrane forms expressed on the surface of HUVEC cells. A Inhibitory effects of G4 and bestatin on the enzymatic activities of different aminopeptidases (rCD13, APA, and APB). Various amounts of G4 or bestatin were incubated with rCD13 (0.2 µg/mL in 60 mM Tris–HCl pH 7.4), APA (0.2 µg/mL in 60 mM Tris–HCl pH 7.4, 50 mM calcium chloride, 200 mM sodium chloride) or APB (0.2 µg/mL in 60 mM Tris–HCl pH 7.4, 100 mM potassium chloride) and the corresponding substrates at 0.1 mM ( l -alanine-7-amido-4-methylcoumarin for rCD13, l -glutamic acid γ-(7-amido-4-methylcoumarin) for APA, and l -arginine-7-amido-4-methylcoumarin for APB). The mixtures were incubated for 30 min at 37 °C, and the cleavage of each substrate was monitored by measuring the fluorescence of methylcoumarin ( λ ex 341 nm; λ em 441 nm) using an Infinite 200 PRO plate reader (Tecan). Three independent experiments were performed for each enzyme. The results of one representative experiment for each enzyme are shown (mean ± SEM, duplicates). B Inhibitory effects of G4 and bestatin on soluble CD13 immunocaptured from human serum (sCD13) using microtiter plates coated with anti-CD13 mAb WM15. The microtiter plate was filled with a solution of mAb WM15 (5 μg/ml in DPBS) and incubated overnight at 4 °C. The plate was then washed with DPBS, blocked with 3% bovine serum albumin (BSA) in DPBS, and filled with human serum containing soluble CD13 (diluted 1:2 with DPBS containing 2% BSA). After washing with DPBS, mixtures of G4 or bestatin (0–100 µM in 60 mM Tris–HCl, pH 7.4) and l -alanine p-nitroanilide hydrochloride substrate (4.5 mM in 60 mM Tris–HCl, pH 7.4) were added and left to incubate at 37 °C for 2 h. Substrate cleavage by sCD13 dissociated from mAb WM15 was then monitored by measuring the absorbance at 405 nm in each well using a plate reader (Bio-Rad). The results of one experiment performed in triplicate are shown (mean ± SEM). C Effect of G4 on the binding of the anti-CD13 mAb WM15 to HUVEC. HUVEC were seeded at a density of 2 × 10 5 cells in 100 µL DPBS supplemented with 5% normal human serum (NHS) and incubated with a mixture of G4 peptide (0, 0.1, 1, and 10 µM) and mAb WM15 (0 or 0.13 nM) as indicated. As a negative control, we used the 11-mer peptide CgA 429–439 , which is a human chromogranin A fragment. Cells were incubated for 60 min at 4 °C, washed, and resuspended in 100 µL of DPBS containing 5% NHS and 1 µg/mL FITC-labeled goat anti-mouse IgG secondary antibody. After incubation for 60 min at 4 °C, the cells were washed and analyzed by flow cytometry. Data are expressed as the percentage of counts vs. fluorescence intensity units
Article Snippet: Phenyl-Sepharose 6 Fast Flow (cat. 17097305), DEAE-Sepharose Fast Flow (cat. 17070901), and Sephacryl-S 300 HR (cat. 17059901) were obtained from Cytiva. l -alanine 7-amido-4-methylcoumarin (cat. 4028736), l -glutamic acid γ- (7-amido-4-methylcoumarin) (cat. 4002702), and l -arginine-7-amido-4-methylcoumarin (cat. 4002148) were obtained from Bachem; His-tagged recombinant human CD13 (rCD13) (cat. 10051-H08H) consisting of the CD13 extracellular domain expressed in HEK-293 cells, His-tagged recombinant human aminopeptidase A (APA) (cat. 10554-H07B) and rabbit anti-human CD13 polyclonal antibody (cat. 10051-RP02) were purchased from Sino Biological.
Techniques: Membrane, Incubation, Fluorescence, Binding Assay, Negative Control, Labeling, Flow Cytometry
ABclonal Biotechnology is a verified supplier 
